Covalent binding of benzo[a]pyrene to DNA in fish liver

Vol. 103, No. 2,19Bl

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

30,1981

November

pages

COVALENT

Usha

BINUiNG DNA IN

Varanasi,

trF BENZUlajPYRENE FISH LIVER

John

E.

Stein

Received

October

TO

and

Northwest and Alaska Fisheries Environmental Conservation 2725 Montlake Boulevard Seattle, Washington

780-787

Tom Horn

Center, Uivision East

NMFS

98112

21,1981

SUMMARY: BenzoLaApyrene became bound to the hepatic DNA in juvenile English sole (Paro hr s vetulus) force fed tritiated benzolajpyrene. No statistical +- y slgniflmchange was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the nurnber of benzo[a]pyrene molecules bound per 106 nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary glanu in which benzo[ajpyrene is carcinogenic. INTHUDUCTIUN:

Bottom-dwelling

pleuronectid

family,

can

(1).

English

sole,

areas

exhibit

a high

possible

accumulate

them

extensively

mutagens

(5)

liver

of

whether

of

BaP-exposed

(e.g.,

liver

BaP)

half-life

levels

of

from

diet

of

sole of to

belonging PAHs

(3-4).

(2)

interact

their

The in

polluted

cellular

a

(1). and

English metabolize

characterized (6)

important fish

the environment

indicating

sediment

mammals

with

in

development and

formed

to

chemically

BaP metabolites in

UaP

from

tumor

carcinogens

English

those

neoplasms

with

A number

proximate

as

sampled of

metabolites long

high

pollutants

(3-4).

reactive

sufficiently

encounter

frequency

PAHs

and

such

a pleuronectid,

association

sole

fish,

are

as

formed

in

question liver

have

was a

macromolecules,

Abbrevidtions used: PAH, polynuclear aromatic hydrocarbon; BaP, benzo[aJpyrene; DMBA, 7,12-dimethylbenzlajanthracene; SDS, sodium dodecylsulfate; IxSSC, 0.15 2 sodium chloride, U.Ulb M sodium citrate, pH 7.4; CBI, covalent binding index; BPDE, (t)-78 , 8-(x -dihydroxy-9 10a -epoxy-7,8,Y,l&tetrahydroxybenzoLajpyreTie.

does

Uisclaimer: not constitute

Mention of endorsement

trade by

names is the U.S.

0006-291X/81/220780-08$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduciion in any form reserved.

780

for information Uepartment of

only and Commerce.

,

Vol. 103, No. 2,Wl

especially cancer that

BIOCHEMICAL

DNA, because through

with

binding

comparative

data

to that In this

BIOPHYSICAL

believed

with

of binding

the

of --in vivo

similar

is

interaction

the magnitude

roughly

it

AND

DNA.

that

chemical

carcinogens

induce

Studies

with

mammals (6-11)

show

of carcinogens

carcinogenic

potential

whether

the extent

paper

we provide

information to hepatic

expose;

to orally

BaP.

the chemical least

administered

of BaP to DNA in fish

mammalian

provide

correlates Determination

valuable

of binding

in fish

is

in mammals.

of BaP intermediate(s)

for

vivo

of the compound.

binding

of binding

to DNA --in

of PAHs, such as BaP, should to assess

RESEARCH COMMUNICATIONS

tissues

susceptible

modification

2 wk after

a single

of the exposure

on the extent DNA and protein

The results

liver

show that

was comparable

to PA&induced hepatic

of covalent in

English

the extent

to values

neoplasms

DNA was observed

sole

reported

and that for

at

to BaP.

MATERIALS AND METHODS: Sodium p-aminosalicylate, deproteinized salmon speml DNA, SDS, ribonuclease-A type l-A, and protease type V were purchased from Sigma Chemical Co., St. Louis, Missouri. The ribonuclease was dissolved in water (1 my/ml) and heated at 80-85°C for 15 min to inactivate any contaminating deoxyribonucleases; the protease was also dissolved in water (1 mg/ml), self-digested for 1 h at 37"C, heated for 2 min at 80°C then quenched in ice. The ribonuclease and protease solutions were stored at -2U'C. Generally labeled [3H]-BaP was purchased from Amersham-Searle, Arlington Heights, Illinois. The [3H]-BaP was purified as described previously (12). The m-cresol was distilled under reduced pressure. All other chemicals were of reagent or analytical grade and used without further purification. Animals: English sole (118+19g), obtained by trawling from Puget Sound, were kept in flowing seawater at 11°C and fed a diet of minced clams for three weeks. The feeding was stopped two days prior to starting the experiment and resumed 48 h after initiation of exposure, with the fish being fed every other day. The fish were force-fed gelatin capsules (no. 3) containing 0.69 mCi of L3H]-BaP (sp act 17.4 Ci/mmole) dissolved in 50 vl of corn oil. The average dose of BaP was 88t12 Pg/kg body weight. Fish were sacrificed by a blow to the head at-16, 24, 96, 168 and 336 h after being fed BaP; the liver was removed and stored at -65'C. The concentration of radioactivity in liver Radioactivity in Liver: was determined from a sample (O.lg) of liver solubilized in 1 ml of Soluene-350 (Packard Instrument Co.) and decolorized with 200 1~1 of both isopropanol and 30% hydrogen peroxide; 15 ml of Dimilume-30 (Packard Instrument Co.) was added and the radioactivity measured using a Packard The distribution of radioactivity 300C liquid scintillation spectrometer.

781

Vol. 103, No. 2.1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

in liver as unconverted BaP, organic solvent-soluble BaP metabolites, and water-soluble BaP metabolites was determined by homogenization of liver and ethyl acetate extraction, followed by thin-layer chromatography A 200 !~l aliquot of the liver homogenate was of the organic phase (4). diluted to 1 ml with water, and an aliquot removed for measurement of The remaining aqueous solution was mixed with 1 volume radioactivity. of N2-saturated acetone, then extracted twice with 1 volume N2-saturated ethyl acetate. The organic extract waS dried over Na2S04, an aliquot removed and radioactivity measured. The remaining extract was removed by suction, evaporated to dryness under a stream of nitrogen, then dissolved in 50-100 Ul of ethyl acetate containing BaP and its oxygenated standards. The organic extract with added standards was spotted on the pre-adsorbent layer of a channeled thin-layer chromatographic plate (Whatman No. LKSU, Whatman Inc., Clitton, N.J.) and developed with hexane: ether (Y5:5, v/v). The adsorbent from the thin-layer chromatoyraphic plate was removed in three fractions. The first fraction contained BaP metabolites (Rf = 0.1) and the pre-adsorbent pad; the second fraction was the adsorbent between Rf of 0.1 and 0.5. The third fraction of adsorbent contained unmetabolized BaP (Rf = 0.5). The radioactivity was measured in each fraction. All of these operations were performed under red light to minimize photooxidation.

Isolation of Liver DNA: Liver DNA was isolated according to parts of the methods by Kirby (13), Kirby and Cook (14) and Marmur (15): Liver tissue (l-29) was minced in 5 volumes of sodium p-aminosalicylate, set-butanol, tetrasodium EDTA dihydrate, water (1:1:0.35:143 w/w/w/v) adjusted to pH 8 with hydrochloric acid. SDS was added to a final concentration of 1% just prior to homogenization in a loose-fitting Teflon-glass homogenizer. The homogenization was performed at 0°C. The homogenate was gently shaken with an equal volume of phenol reagent (phenol, m-cresol, &hydroxyquinoline, water lOU:l4:U.l:11 w/w/w/w) After centrifugation for 40 min at 4'C and 10,OOOxg for 45 min at 40°C. the aqueous phase was removed by suction. The phenol reagent fraction was re-extracted with 0.5 volume of the sodium p-aminosalicylate mixture. NaCl was added to the combined aqueous phases to a final concentration of 3% (w/v) and shaken with 0.5 volume of phenol reagent for 10 min and centrifuged as described above. The phenol reagent fractions were combined and stored at -20% for later protein isolation. The viscous aqueous phase was shaken for 5 min with 1 volume of chloroform-isoamyl alcohol (24:l v/v) and centrifuged at 3UUOxg for 5 min. Crude DNA was precipitated by addition of 2 volumes of 2-ethoxyethanol (-2O'C) and spooled onto a glass rod. The nucleic acids were dissolved overnight at 4'C in 5-10 ml of O.lxSSC, and after dissolution the solution was were degraded adjusted to 1xSSC by adding 1OxSSC. RNA contaminants by a 1 h incubation at 37°C with ribonuclease solution (0.1 ml, 1 my/ml). Protein contaminants were rernoved by addition of protease (0.1 ml, 1 mg/ml) and 0.25 volumes of 20% SDS to bring the solution to 0.5% SOS, followed by overnight incubation at 37°C. SDS was added to a final concentration of l%, and the DNA solution was extracted with 1 volume of chloroform-isoamyl alcohol; the DNA was precipitated with 2 volumes of ethanol (-2VC). The DNA was washed twice with ethanol, once with ether and dryed under a stream of N2. The concentration of DNA, dissolved in 2 ml of deionized distilled water, was estimated from the absorbance at 260 nm using an extinction coefficient of 22.Y ml/mg-cm, which was determined for salmon sperm DNA. DNA isolated by this procedure had A260:&0 and A26O:A230 values of 1.82tU.03 and 2.2+0.2, (10). Prior to liquid scintillation countTng the DNA . respectively solution was made U.5N in perchloric acid using concentrated perchloric

782

Vol. 103, No. 2,198l

TABLE

1.

Exposure, h

Distribution metabolites 3H-BaP.

BIOCHEMICAL

of BaP as parent to DNA and protein

% Administered Dose

hydrocarbon in liver

(n=4)e 0.6+o.lf

24

BIOPHYSICAL

or of

Organic Soluble Metabolites

Unconverted BaP

% of Total

16

AND

metabolites English sole

Water Soluble Metabolites

RESEARCH COMMUNICATIONS

and after

extent of exposure

Specifica Activity (Protein)

51+31 -

45226

16210

56+39

42t15

46218

135

95 92

14t - 2

25+18

22216

86 95

31518

50232

46228

1.1 0.3

9.5 12

89 88

90224

96

U.4zD.l

0.2 0.7

96 93

168

u.2+0.1

0.4 0.7

4.8 7.6

0.7 0.4

11 4.3

fmole BaP equivalents/mg DNA or protein. pm administered/kg fish)j/3.24x10-g=(lle CBIPM 7 L(dpm/mgDNA)/(d nut eotides)/(mmole BP administered/kg fish); using 3099 nrole BaP equivalents/mole nucleotides. Since only two fish were analyzed, individual values are Number of fish analyzed at each time point. Mean + standard error.

DNA=1

mole

BP equivalents/ of nucleotides.

mole

given.

and heated for 20 min at 1UU"C. Insta-gel (Packard was added and the radioactivity was measured.

Instrument

Isolation of Liver Protein: The phenol reagent phases were treated essentially according to method (b) of Poland and tilover (161, except the protein was precipitated by the addition of two volum& bf acetone followed by the use of 10% SUS in both dissolution-precipitation steps. The final precipitate was washed, in centrifuge tubes, twice with acetone, once with ether and dryed under a stream of nitrogen. An aliquot (20 mg) was first dissolved in 1 ml of Soluene-350 then 15 ml of Uimilume-30 was added and the radioactivity measured. All data were analyzed by one-way analysis of Statistical Analysis: variance. The averase of all time periods for specific activities for protein and DNA were tested for'difference uiing Students t-test and the Mann-Nhitney test, at a significance level of 0.05. RESULTS:

Kadioactivity

in liver

rePresented

dose at 16 h (Table

1).

The concentration

in

sole

did

liver

of English

336 h after

the

total

radioactivity

oral

not

administration in liver

Bindingc Values

(n=3) 165 9f

o.eo.2

acid Co.)

CBIb

(n=3) 412251

83 a4

c d 'f

Specifica Activity

DNA

16 15

i

binding of administered

Radioactivityd

0.8 1.2

336

covalent to orally

change

0.6% of the of BaP-derived

significantly

of BaP; greater

was in the

783

administered

form

radioactivity

from than

of the organic

16 h through

98% of the solvent-

5

85 6 16+10

Vol. 103, No. 2,198l

BIOCHEMICAL

and water-soluble the

organic

because

metabolites

or aqueous

detailed

metabolites

data

expressed

to DNA in liver

for

extent

of binding the

different

the

(Table for

lesions

The results

DNA in English

since

these

PAHs (l-10 liver

did

BaP

(pmole

body weight), the

to

data

are

of BaP intermediates BaP-exposure.

1).

As with

not

change

DNA, the

significantly of all

the

was not significantly

DNA, 44239;

individual

In

in the binding

the average

58251,

for

for

to hepatic

of activated

sole

the

the

values

values

for

first

time

are

15 fish

expressed analyzed

findings

exposed

the --in vivo

macromolecules,

carcinogens

implicated

do encounter

ppm) in their

neoplasia

indices

was observed

Moreover,

protein,

(nmole

mammals where

(Table

protein

demonstrate

The present

liver

fish

change

from

DNA, is strongly (6-11).

binding

activities

experiment.

Interaction

especially

or sediment

values

16 to 336 h after

value

of BaP intermediate(s)

in fish.

from

1).

as the mean + SD calculated

binding

diet

as specific

binding

2 wk exposure

from the average

DISCUSSION:

study

modes.

detectable

of BaP to liver

the entire

for

three

significant

values

both

binding

data

1 show that

activity

in

and conjugated

DNA are given

and covalent

in one of these

2 wk period

specific

during

1) for

published

DNA during

in this

BaP administered/kg

was observed

values

to BaP via

macromolecule),

no statistically

metabolites

not characterized

exposed

(Table

with

Data in Table

Individual

of nonconjugated

of nucleotides)/(mmole

usually

fact,

were

of nucleotide)

comparison

1).

RESEARCH COMMUNICATIONS

(3-4).

BaP equivalents/mg

permit

over

sole

earlier

The binding

BaP/mole

(Table

phases

in English

equivalent/mole

BIOPHYSICAL

characterization

has been published

(fmole

AND

with

macromolecules,

in initiation showing

DNA and protein,

of malignant

chemical

modification

to BaP is of considerable

high

levels

environment

(1)

(2).

784

of BaP and other and they

are

of

importance carcinogenic

susceptible

to

Vol.103.No.2,1981

BIOCHEMICAL

In a separate simultaneously

experiment

to L14C]

injection.

indicating

tritiated

that

of labeled

BaP fragments

of a single

for

per

body weight

mammalian

mammals,

Lutz

and 4 for

liver

of rats

gland

a Cl31 of 24 for

are susceptible

a greater

extent

rats

than

(20)

susceptible

at any time

In the during

tissues

susceptible

between

covalent

tissue

tissue

the

binding

carcinogen

(19).

range

valid,

then

English

785

a CBI of 56

were

Both

sole

feeding

obtained

when

and mammary DMBA binds

of partially liver

while

liver

for

of values Thus,

results liver.

rapidly

rodents

intact

value

to

hepatectomized

(CBI=4);

DNA and the our

a single

skin

(7-11).

neoplasms.

of a PAH with

of our data

the dose of PAH

hepatectomized (21)

administration

comparison

liver

the average

2 wk was in the

for

deoxyadenosine

after

at 16 h after

adult

neoplasms

is

a rat

incorporation

calculated

(CBI=37)

intact

study

(10)

neoplasia

to PAH-induced

to the carcinogen

is a potential

route

of partially

present

performed

Based on a compilation

and 7 for

liver

DNA from

to DMBA-induced

is not.

skin oral

to DNA from

liver

studies.

to PAH-induced

to the

proliferating

rat

to BaP via

and

shows how many molecules

although

with

exposed

unaltered

allows

tissues

studies

mice were

with

(17)

DNA from

106 DNA nucleotides

of data

of DMBA (18);

et al. liver

as Cl31 which

in various

mammary gland

[I4C]

DNA using

no biosynthetic

is different

for

both with

Viviani

associated

administered from

for

of UNA by l3aP intermediates

digested

expressed

covalently

reported

exposed

DNA.

dose of 1 mmole/kg

those

DNA.

were

an intraperitoneal

associated

practically into

potency

of a PAH are bound

with

fish

similar

modification

no tritium

indicating

The binding

DNA were

of enzymatically

and found

or deoxyguanosine

BaP via

radioactivity

exchange

analysis

[3H]-BaP,

with

the

RESEARCH COMMUNICATIONS

to be published)

for

BaP was due to chemical

a nucleoside fed

(data

values

and not due to tritium

BIOPHYSICAL

and C3H]-labeled

The binding

L3H]-BaP

AND

of

CBI (Table for

if

is

1)

mammalian

a correlation

susceptibility suggest

of a that

BaP

Vol.103,No.2,1981

BIOCHEMICAL

For at least liver

remained

studies

with

binding,

the

tissues

also

2 wk after

chemically

the

persistence

in

regenerating

liver

of DMBA, while

observed

liver.

within

constant the

rat

level

PAH.

its

binding, a long

is

ACKNOWLEDGEMENTS: Protection

Agency

by increased cells This

that

DMBA-DNA at a relatively

administration

slow

lipid

this

release

(27),

(30)

observed of sister

BaP-exposed

was supported

in part

of PAH or with of

modified

for

may adversely

et al. frequency

might

persistence

remained

interaction

of

coupled

of the

such as replication

from

was not

of a PAN-DNA complex

liver

DMBA-DNA

4 wk after

was maintained

or the

to

that

binding

events

isolated work

at least

of the cause

because

Stromberg

reported

intragastric

DNA in sole

of critical

(29).

in kidney

that

(25)

observed

cells

intracellular

Regardless

important,

a number

indicated

(26)

mechanism

from

activation.

transcription

rates,

repair

the observation

influence

damage,

a slow

of the tissue

for

or maintenance

metabolites

period

et al.

3 to 14 days after

The persistence

primary

subsequent

Janss

of

of DNA in target

such persistent

mammary parenchymal

from

be due to either

et al.

persists

from

to the magnitude

on susceptibility

the administration

complex

Evidence

modification

Marguardt rat

to BaP, DNA in sole

level.

in addition

information

RESEARCH COMMUNICATIONS

exposure

at a high

that

(Z-25).

in intact

single

of chemical

provides lesions

BIOPHYSICAL

modified

mammals suggests

neoplastic complex

AND

(28)

significant chromatid

English by the

and chromosomal exchange

sole. Environmental

(EPA 79-D-X0514).

HEFERENCES 1. 2. 3. 4.

Malins, D.C., McCain, B.B., Brown, D.W., Sparks, A.K. and Hodgins, H.O. (1980) NOAA Tech. Memo. OMPA-2, pp l-295. Pierce, K.V., McCain, B.B. and Wellings, S.R. (1978) J. Natl. Cancer Inst., 6J, 1445-1453. Varanasi, U. and (;mur, D.J. (1981) Chemical Analysis and Biological Fate: Polynuclear Aromatic Hydrocarbons (Cooke, M. and Dennis, A.J. eds), pp 367-376, Battelle Press, Columbus. Varanasi, U. and Gmur, D.J. (1981) Aquatic Toxicol., -,1 49-67.

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Vol. 103, No. 2,198l

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

T. (1978) Polycyclic Hydrocarbons and Cancer, Nagao, M., and Sugimara, Vol. 2 (tielboin, H.V., and Ts'lJ, P.O.P., eds), pp. 99-121, Academic Press, New York. Kapitulnik, J., Levin, W., Conney, A.H., Yagi, H. and Jerina, D.M. (1977) Nature, 266, 378-380. Brooks, P. and Lxey, P.D. (lY64) Nature, 202, 781-784. Miller, E.C. and Miller, J.A. (lY74) The Molecular Biology of Cancer ('Busch H, ed.), pp. 377-402, Academic Press, London. Huberman, E. and Sachs, L. (1977) Int. J. Cancer, 19, 122-127. Lutz, W.K. (1979) Mutation Res., 65, 289-356. Phillips, U.H., Grover, P.L., and gms, P. (1979) Int. J. Cancer, g, ml-208. Varanasi, IJ. and Gmur, D.J. (1980) Biochem. Pharniacol., 2, 753-761. Kirby, K.S. (1965) Biochem. J., yd, 266-269. Kirby, K.S. and Cook, E.A. (1967) Biochem. J., 104, 254-257. Marmur, J. (1961) J. Mol. Biol., 3, 208-218. Poland, A. and Glover, E. (lY7q Cancer Res., 3y, 3341-3344. Viviani, A., von Daniken, A., Schlatter, Ch. and Lutz, W.K. (1980) J. Cancer Res. Clin. Oncol., 98, 139-152. Janss, D.H. and Ben, T.L. (19n) J. Natl. Cancer Inst., so, 173-177. Suss, K. and Maurer, H.R. (1968) Nature, 217, /52-753. Marquardt. H.A., Bendick, A., Philips,F.S. and Hoffmann (1971) Chem.-Viol. Interact., 3, l-11. Marquardt, H., Sternberg, S.S. and Philips, F.S. (1970) Chem.-Biol. Interact., -,2 4Dl-4U3. Lo, K. and Kakunaga, T. (1981) Biochem. Biophys. Res. Commun., ,U, 820-829. Margison, G.P. and Kleihaus, P. (1975) Biochem J., 148, 521-525. Kleihaus, P. and Margison, G.P. (lY74) J. Natl. Cancer Inst., 53, 183Y-1841. Marquardt, H.A., Bendick, A. and Philips, F.S. (1971) Proc. Am. Assoc. Cancer Kes., lZ-, 15. Janss, D.H., Moon K.C. and Irving, C.C. (1972) Cancer Res., 32, 254-258. Wills, E.U. (1980) Microsomes, Drug Oxidations, and Chemical Carcinogenesis(Coon, M.J ., Conney, A.H., Estabrook, K.W., J.R. and O'Brien, P.J. eds), pp 545-548, tielboin, H.V., Gillette, Academic Press, New York. Lin, E.J. and Weiss, S.B. (1977) Proc. Natl. HSU, W.T., Harvey, K.ti., Acad. Sci. U.S.A., 74, 1378-1382. Leffler, S., PulkraEk, P., Grunberger, D. and Weinstein, I.B. (lY77) Biochemistry, 2, 3133-3136. Stromberg, P.T., Landolt, M.L. dnd Kocan, K.M. (1981) NOAA Tech Memo. DMPA-10, pp l-43.

787